Download Myco-TB – TB IgG

Myco-TB – TB IgG
Catalog #: WT1096
IgG ANTIBODIES TO M. TUBERCULOSIS ELISA KIT
Two-Step Incubation, Indirect Principle
INSTRUCTIONS FOR USE
This kit is an enzyme-linked immunosorbent assay for
qualitative detection of antibodies to Syphilis in human
serum or plasma. For research use only.
SUMMARY
It has been estimated that more than 1.7 million people
were infected with Mycobacterium tuberculosis (MTb) in
1990 and this number increase every year due to new
infections (HIV) and immigration. Now, with worldwide
prevalence of 30 million cases and an incidence of 10
million new cases each year, tuberculosis (TB) is one of
the most important health threatening problems worldwide
PRINCIPLE OF THE ASSAY
This kit employs solid phase, indirect ELISA assay for
detection of IgG antibodies to MTb in two-step incubation
procedure. Polystyrene microwell strips are pre-coated
with affinity purified MTb antigens including A60 antigen
complex. During the first incubation step, MTb IgG specific
antibodies, if present, will be bound to the solid phase precoated antigen complexes. The wells are washed to
remove unbound serum proteins, and rabbit anti-human
IgG antibodies (anti-IgG) conjugated to horseradish
peroxidase (HRP) are added. During the second
incubation step, these HRP-conjugated antibodies will be
bound to any antigen-IgG complexes previously formed
and the unbound HRP-conjugate is then removed by
washing.
Chromogen
solutions
containing
Tetramethylbenzidine (TMB) and urea peroxide are added
to the wells.
In presence of the antigen-(IgG)-anti-IgG (HRP)
immunocomplex,
the
colorless
Chromogens
are
hydrolyzed by the bound HRP conjugate to a blue colored
product. The blue color turns yellow after stopping the
reaction with sulfuric acid.
The amount of color intensity can be measured and is
proportional to the amount of antibody captured in the
wells, and to the sample respectively. Wells containing
samples negative for IgG antibodies to MTb remain
colorless.
COMPONENTS

MICROWELL PLATE
1 plate
Blank microwell strips fixed on white strip holder. The plate
is sealed in aluminum pouch with desiccant.
8×12/12×8-well strips per plate.
Each well contains affinity purified MTb antigens. The
microwell strips can be broken to be used separately.
Place unused wells in the plastic sealable storage bag together
with the desiccant and return at 2~8ºC.

NEGATIVE CONTROL
1 vial
Colorless liquid filled in vial with green screw cap
0.5ml per vial.
Protein-stabilized buffer tested nonreactive for MTb IgG antibodies. Preservatives: 0.1%
ProClin 300. Ready to use as supplied. Once open,
stable for one month at 2-8ºC.

POSITIVE CONTROL SERUM
1 vial
Red liquid filled in vial with red screw cap
0.5ml per vial. MTb antibodies diluted in protein-stabilized
buffer containing preservatives: 0.1% ProClin 300. Ready
to use as supplied. Once open, stable for one month at 28ºC.

SPECIMEN DILUENT
1 vial
Blue liquid filled in a white vial with blue screw cap. 12 ml
per vial Protein-stabilized buffer, casein, and sucrose
solution. Ready to use as supplied. Once open, stable for
one month at 2-8ºC.

HRP-CONJUGATE REAGENT
1 vial
Red liquid filled in a white vial with red screw cap.
12ml per vial. Horseradish peroxidase-conjugated rabbit
anti-human IgG antibodies. Ready to use as supplied.
Once open, stable for one month at 2-8°C.

STOCK WASH BUFFER
1 bottle
DILUTE BEFORE USE
Colorless liquid. 50 ml per bottle. PH 7.4, 20 × PBS
(Containing Tween 20 as a detergent) The concentrate
must be diluted 1:20 with distilled/deionized water before
use. Once diluted, stable for one weeks at room temperature
or for two weeks at 2-8ºC.

CHROMOGEN SOLUTION A
1 vial
Colorless liquid filled in white vial with green screw cap.
6 ml per vial. Urea peroxide solution. Ready to use as
supplied. Once open, stable for one month at 2-8ºC.

CHROMOGEN SOLUTION B
1 vial
Colorless liquid filled in the black vial with black screw cap.
6 ml per vial. TMB solution- Tetramethylbenzidine
dissolved in citric acid. Ready to use as supplied.
Once open, stable for one month at 2-8ºC.

STOP SOLUTION
1 vial
Colorless liquid filled in white vial with yellow screw cap.
6 ml per vial. Diluted sulfuric acid solution (2.0M H2SO4).
Ready to use as supplied.

PLASTIC SEALABLE BAG
1 unit
For enclosing the strips not in use.
 CARDBOARD PLATE COVER
2 sheets
To cover the plates during incubation and prevent evaporation
or contamination of the wells.
 PACKAGE INSERTS
1 copy
ADDITIONAL MATERIALS AND INSTRUMENTS
REQUIRED BUT NOT PROVIDED
1.
2.
3.
Freshly distilled or deionized water.
Disposable gloves and timer.
Appropriate waste containers for
potentially
contaminated materials.
4.
Disposable V-shaped troughs.
5.
Dispensing system and/or pipette (single or
multichannel), disposable pipette tips.
6.
Absorbent tissue or clean towel.
7.
Dry incubator or water bath, 37±0.5°C.
8.
Microshaker for dissolving and mixing conjugate with
samples.
9.
Microwell plate reader, single wavelength 450nm or
dual wavelength 450nm and 630nm.
10. Microwell aspiration/wash system.
5.
6.
7.
SPECIAMEN COLLECTION, TRANSPORTATION
AND STORAGE
1.
2.
Sample Collection: Either fresh serum or plasma
samples can be used for this assay. Blood collected
by venipuncture should be allowed to clot naturally
and completely – the serum/plasma must be
separated from the clot as early as possible as to
avoid hemolysis of the RBC. Care should be taken to
ensure that the serum samples are clear and not
contaminated by microorganisms. Any visible
particulate matters in the sample should be removed
by centrifugation at 3000 RPM for at least 20 minutes
at room temperature, or by filtration on 0.22u filters.
Plasma samples collected into EDTA, sodium citrate
or heparin may be tested, but highly lipaemic, icteric,
or hemolized samples should not be used as they
could give erroneous results in the assay. Do not
heat inactivate samples. This can cause sample
deterioration.
Transportation and Storage: Store samples at 28℃. Samples not required for assaying within 3 days
should be stored frozen (-20°C or lower).Multiple
freeze-thaw cycles should be avoided. For shipment,
samples should be packaged and labeled in
accordance with the existing local and international
regulations for transport of clinical samples and
ethological agents.
STORAGE AND STABILITY
The components of the kit will remain stable through the
expiration date indicated on the label and package when
stored between 2-8°C, do not freeze. To assure
maximum performance of this TB IgG ELISA kit, protect
the reagents from contamination with microorganism or
chemicals during storage.
PRECAUTIONS AND SAFETY
This kit is intended FOR RESEARCH USE ONLY
The ELISA assay is a time and temperature sensitive
method. To avoid incorrect result, strictly follow the test
procedure steps and do not modify them.
1.
2.
3.
SPECIAL INSTRUCTIONS FOR WASHING
1.
2.
3.
4.
A good washing procedure is essential to obtain
correct and precise analytical data.
It is therefore recommended to use a good quality
ELISA microplate washer, maintained at the best
level of washing performances. In general, no less
than 5 automatic washing cycles with dispensing of
350-400µl/well, are sufficient to avoid false positive
reactions and high background (all wells turn yellow).
To avoid cross-contaminations of the plate with
sample or HRP-conjugate, after incubation do not
discard the content of the wells, but allow the plate
washer to aspirate it automatically.
Anyway, we recommend calibrating the washing
system on the kit itself in order to match the declared
analytical performances. Assure that the microplate
washer’s liquid dispensing channels are not blocked
or contaminated, and sufficient volume of Wash
buffer is dispensed each time into the wells.
In case of manual washing, we suggest to perform at
least 5 cycles, dispensing 350-400µl/well and
aspirating the liquid for 5times. If poor results (high
background) are observed, increase the washing
cycles or soaking time per well.
In any case, the liquid aspirated out the strips should
be treated with a sodium hypochlorite solution(final
concentration of 2.5%) for 24 hours, before liquids
are disposed in an appropriate way.
The concentrated Washing solution should be diluted
1 to 20 before use. For one plate, mix 50 ml of the
concentrate with 950ml of water for a final volume of
1000ml diluted Wash Buffer. If less than a whole
plate is used, prepare the proportional volume of
solution.
4.
5.
6.
7.
8.
9.
Do not exchange reagents from different lots, or use
reagents from other commercially available kits. The
components of the kit are precisely matched as to
achieve optimal performance during testing.
Make sure that all reagents are within the validity
indicated on the kit box and are of the same lot.
Never use reagents beyond the expiry date stated
on reagents labels or on the kit box.
CAUTION - CRITICAL STEP: Allow the reagents and
samples to stabilize at room temperature (18-30°C)
before use. Shake reagent gently before, and return
to 2-8°C immediately after use.
Use only sufficient volume of sample as indicated in
the procedure steps. Failure to do so, may cause in
low sensitivity of the assay.
Do not touch the bottom exterior of the wells;
fingerprints or scratches may interfere with microwell
reading.
When reading the results, ensure that the plate
bottom is dry and there are no air-bubbles inside the
wells.
Never allow the microplate wells to dry after the
washing step. Immediately proceed to the next step.
Avoid the formation of air-bubbles when adding the
reagents.
Avoid assay steps long time interruptions. Assure
same working conditions for all wells.
Calibrate the pipette frequently to assure the
accuracy of samples/reagents dispensing. Always
use different disposal pipette tips for each specimen
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
and reagents as to avoid cross-contaminations.
Never pipette solutions by mouth.
The use of automatic pipettes is recommended.
Assure that the incubation temperature is 37°C
inside the incubator.
When adding samples, avoid touching the well’s
bottom with the pipette tip.
When reading the results with a plate reader, it is
recommended to determine the absorbance at
450nm or at 450nm with reference at 630nm.
All specimens from human origin should be
considered as potentially infectious.
Materials from human origin may have been used in
the kit. These materials have been tested with tests
kits with accepted performance and found negative
for antibodies to HIV ½, HCV, TP and HBsAg.
However, there is no analytical method that can
assure that infectious agents in the specimens or
reagents are completely absent. Therefore, handle
reagents and specimens with extreme caution as if
capable of transmitting infectious diseases. Strict
adherence to GLP (Good Laboratory Practice)
regulations can ensure the personal safety. Never
eat, drink, smoke, or apply cosmetics in the assay
laboratory.
Bovine derived sera may have been used in this kit.
Bovine serum albumin (BSA) and fetal calf sera
(FCS) are derived from animals from BSE/TSE freegeographical areas.
The pipette tips, vials, strips and sample containers
should be collected and autoclaved for 1 hour at
121°C or treated with 10% sodium hypochlorite for
30minutes to decontaminate before any further steps
for disposal.
The Stop solution (2M H2SO4) is a strong acid.
Corrosive. Use it with appropriate care. Wipe up
spills immediately or wash with water if come into
contact with the skin or eyes. ProClin 300 used as a
preservative can cause sensation of the skin.
The enzymatic activity of the HRP-conjugate might
be affected from dust, reactive chemical, and
substances like sodium hypochlorite, acids, alkalis
etc. Do not perform the assay in the presence of
such substances.
Materials Safety Data Sheet (MSDS) available upon
request.
If using fully automated microplate processing
system, during incubation, do not cover the plates
with the plate cover. The tapping out of the
remainders inside the plate after washing, can also
be omitted.
Step 1 Reagents preparation: Allow the reagents and
samples to reach room temperature (18-30°C) for at
least 15-30minutes. Check the Wash buffer
concentrate for the presence of salt crystals. If
crystals have formed in the solution, resolubilize by
warming at 37°C until crystals dissolve. Dilute the
stock wash Buffer 1 to 20 with distilled or deionized
water. Use only clean vessels to dilute the Wash
buffer.
Step 2 Numbering Wells: Set the strips needed in strip-holder
and number sufficient number of wells including three
Negative control (e.g. B1, C1, D1), two Positive control
(e.g. E1, F1) and one Blank (A1, Neither samples nor
HRP-Conjugate should be added into the Blank well). If
the results will be determined by using dual wavelength
plate reader, the requirement for use of Blank well
could be omitted. Use only number of strips required for
the test.
Step 3 Adding Diluent: Add 100µl Specimen Diluent into each
well except in the blank.
Step 4 Adding Sample: Add 10µl of Specimen and 10µl of
Positive and Negative controls into their respective
wells.
Note: Use a separate disposal pipette tip for each
specimen, Negative Control and Positive Control as
to avoid cross-contamination. Mix by tapping the
plate gently.
Step 5 Incubating: Cover the plate with the plate cover and
incubate for 30 minutes at 37°C. It is recommended to
use water tank to assure the temperature stability and
humidity during incubation. If dry incubator is used, do
not open the door frequently.
Step 6 Washing: After the end of the incubation, remove and
discard the plate cover. Wash each well 5 times with
diluted Wash buffer. Each time allow the microwells to
soak for 30-60 seconds. After the final washing cycle,
turn the strips plate onto blotting paper or clean towel,
and tap the plate to remove any remainders.
Step 7 Adding HRP-Conjugate: Add 100 µl HRP-Conjugate
to each well except the Blank.
Step 8 HRP-Conjugate Incubating: Cover the plate with the
plate cover and incubate for 30 minutes at 37°C.
Step 9 Washing: After the end of the incubation, remove and
discard the plate cover. Wash each well 5 times with
diluted Wash buffer as in Step 6.
Step 10 Coloring: Dispense 50µl of Chromogen A and 50µl
Chromogen B solution into each well including the
Blank and mix by tapping the plate gently. Incubate the
plate at 37℃ for 15 minutes avoiding light. The
enzymatic reaction between the Chromogen A/B
solutions produces blue color in Positive control and TB
IgG Positive sample wells.
Step 11 Stopping Reaction: Using a multichannel pipette or
manually add 50 µl Stop Solution into each well and
mix by tapping the plate gently. Intensive yellow color
develops in Positive control and TB IgG Positive
sample wells.
Step 12 Measuring the Absorbance: Calibrate the plate
reader with the Blank well and read the absorbance at
450nm. If a dual filter instrument is used, set the
reference wavelength at 630nm. Calculate the Cut-off
value and evaluate the results. (Note: read the
absorbance within 5 minutes after stopping the
reaction)
INTERPERTATION OF RESULTS AND QUALITY
CONTROL
Each microplate should be considered separately when
calculating and interpreting the results, regardless of the
number of plates concurrently processed. The results are
calculated by relating each sample’s optical density (OD)
value to the Cut-off value (C.O.) of the plate. If the Cut-off
reading is based on single filter plate reader, the results
should be calculated by subtracting the Blank well OD
value from the print report values of samples and controls.
In case the reading is based on dual filter plate reader, do
not subtract the Blank well OD from the print report values
of samples and controls.
1. Calculation of Cut-off value (C.O.) = *NC + 0.15
*NC = the mean absorbance value for three negative controls.
Example:
1. Calculation of NC:
Well No.
B1
C1
D1
Negative controls OD value
0.02
0.012
0.016
NC=0.016
2. Calculation of Cut-off (C.O.)= 0.016+0.15= 0.166
If one of the Negative Control values does not meet the
Quality Control Range specifications, it should be
discarded and the mean value is calculated again using
the remaining two values. If more than one control OD
value does not meet the Quality Control Range
specifications, the test is invalid and must be repeated.
2. Quality Control Range.
The test results are valid if the Quality Control criteria are
verified. It is recommended that each laboratory must
establish appropriate quality control system with quality
control material similar to or identical with the patient
sample being analyzed.
1. The OD value of the Blank well, which contains only
Chromogens and Stop solution, is less than 0.080 at
450 nm.
2. The OD value of the Positive control must be equal to
or greater than 0.800 at 450/630nm or at 450nm after
blanking.
3. The OD value of the Negative control must be less than
0.100 at 450/630nm or at 450nm after blanking.
3. Interpretations of results:
(S = the individual absorbance (OD) of each specimen)
Negative Results (S/C.O. <1): samples giving
absorbance less than the Cut-off value are negative for
this assay, which indicates that no IgG antibodies to
Mycobacterium tuberculosis have been detected with
this TB IgG ELISA kit.
Positive Results (S/C.O.≥1) : samples giving an
absorbance greater than or equal to the Cut-off value are
considered initially reactive, which indicates that IgG
antibodies to Mycobacterium tuberculosis have probably
been detected using this TB IgG ELISA kit. Retesting in
duplicates of any initially reactive sample is
recommended. Repeatedly reactive samples can be
considered positive for IgG antibodies to Mycobacteria.
Borderline: (S/C.O. =0.9-1.1) Samples with absorbance
to Cut-off ratio between 0.9 and 1.1 are considered
borderline and retesting of these samples in duplicates is
recommended to confirm the results. Repeatedly positive
samples can be considered positive for antibodies to MTb.
Analytical Specificity:
1. No cross reactivity observed with samples from patients
infected with HAV, HIV, HBV, HCV, CMV, and TP.
2. No interference was observed from rheumatoid factors
up to 2000U/ml.
3. These
assay
performance
characteristics
are
unaffected from elevated concentrations of bilirubin,
hemoglobin, and triolein.
4. Frozen specimens have been tested to check for
interferences due to collection and storage.
LIMITATIONS
1. Non-repeatable positive result may occur due to the
general biological characteristics of the ELISA method.
The assay is designed to achieve very high
performance characteristics of sensitivity and specificity
and the “indirect” model minimizes the unspecific
reactions due to interference with unknown matters in
sample and the anti-human IgG antibodies. Antibodies
may be undetectable during the early stages of the
disease and in some immunosuppressed individuals.
2. Common sources for mistakes are: kits beyond the
expiry date, bad washing procedures, contaminated
reagents, incorrect assay procedure steps, insufficient
aspiration during washing, failure to add samples or
reagents, equipment, timing, volumes, sample nature
and quality.
3. The prevalence of the marker will affect the assay’s
predictive values.
4. If, after retesting of the initially reactive samples, the
assay results are negative , these samples should be
considered as non-repeatable (false positive) and
interpreted as negative. As with many very sensitive
ELISA assays, false positive results can occur due to
the several reasons, most of which are related but not
limited to inadequate washing step.
5. This is a qualitative assay and the results cannot be
used to measure antibodies concentrations.
INDICATIONS OF INSTABILITY OR
DETERIORATION OF THE REAGENTS
1.
2.
Values of the Positive or Negative controls, which are
out of the indicated Quality control range, are indicator
of possible deterioration of the reagents and/or
operator or equipment errors. In such case, the results
should be considered as invalid and the samples must
be retested. In case of constant erroneous results
classified as due to deterioration or instability of the
reagents, immediately substitute the reagents with new
ones.
If after mixing of the Chromogen A and B solutions into
the wells, the, the color of the mixture turns blue within
few minutes, do not continue carrying out the testing
and replace the reagents with fresh ones.
VALIDITY
Please do not use this kit beyond the expiration
indicated on the kit box and reagent labels
REFERENCES:
1. Hasløv, K. Boosting effects in BCG-vaccinated guinea pigs of
tuberculin skin tests on subsequent skin tests and on the in
vitro lymphocyte transformation response. 1983. International
Symposium on BCG Vaccines and Tuberculins, Budapest,
Hungary. Develop. biol. Standard. 58: 517-521 (S. Karger,
Basel. 1986).
2. Zou YL,Zhnag JD, Chen MH,Shi GQ,Prignot J,Cocito C (1194)
Serological analysis of pulmonary and extrapulmonary
tuberculosis with enzyme-linked immunosorbent assay for
anti-A60 immunoglobulings C.I.D. 19: 1084-1091
3. Wirrmann C(1990) Public health applications of serological
tests for tuberculosis :Study of the incidence of inapparent
infectious employees of an alsacian supermarket. Eur J
Epidemol 6:304-308
4. Harboe M (1981) Antigens of PPD ,old tuberculin and
autoclaved M.bovis BCG studied by cross
immunoelectrophoresis .Am Rev Respir
5. Dis 124:80-87
6. Anda Biologicals: Publications
P.O. BOX 458
Thurmont, MD 21788 USA
Tel: 301-228-2444 Fax: 301-560-6570
Toll Free: 888-562-8914
www.xpressbio.com
[email protected]